Dopamine Receptors Dopamine plays a major role in the regulation of cognitive, emotional and behavioral functions abnormalities in its regulation have been implicated in neuropsychiatric and substance use disorders. Dopamine receptors belong to G protein-coupled receptor (GPCR) family, and are implicated in various vital physiological functions. That D3R expression is elevated in response to drugs of abuse, has prompted efforts toward the development of D3R-selective agents for the treatment of drug addiction. Inhibition of D3R may be less prone to causing motor side effects that can result from D2R blockade. SB269652, a bitopic ligand that simultaneously binds both the orthosteric binding site (OBS) and a SBP in both D2R and D3R, was found to be a negative allosteric modulator. Previous studies identified Glu2.65 in the SBP to be a key determinant of both the affinity of SB269652 and the magnitude of its cooperativity with orthosteric ligands. To understand the structural basis of the observed phenotype of E2.65A, we carried out MD simulations with a cumulative length of 77 s of D2R and D3R wild-type and their E2.65A mutants bound to SB269652. In combination with Markov state model analysis and by characterizing the equilibria of ligand binding modes in different conditions, we found that in both D2R and D3R, whereas the tetrahydroisoquinoline moiety of SB269652 is stably bound in the OBS, the indole-2-carboxamide moiety is dynamic and only intermittently forms H-bonds with Glu2.65. Our results also indicate that the E2.65A mutation significantly affects the overall shape and size of the SBP, as well as the conformation of the N terminus. Thus, our findings suggest that the key role of Glu2.65 in mediating the allosteric properties of SB269652 extends beyond a direct interaction with SB269652, and provide structural insights for rational design of SB269652 derivatives that may retain its allosteric properties. The two highly homologous subtypes of stimulatory G proteins Gs (Gs) and Golf (Golf) display contrasting expression patterns in the brain. Golf is predominant in the striatum, while Gs is predominant in the cortex. Yet, little is known about their functional distinctions. The dopamine D1 receptor (D1R) couples to Gs/olf and is highly expressed in cortical and striatal areas, making it an important therapeutic target for neuropsychiatric disorders. Using novel drug screening methods that allow analysis of specific G-protein subtype coupling, we found that, relative to dopamine, dihydrexidine and N-propyl-apomorphine behave as full D1R agonists when coupled to Gs, but as partial D1R agonists when coupled to Golf. The Gs/Golf-dependent biased agonism by dihydrexidine was consistently observed at the levels of cellular signaling, neuronal function, and behavior. Our findings of Gs/Golf-dependent functional selectivity in D1R ligands open a new avenue for the treatment of cortex-specific or striatum-specific neuropsychiatric dysfunction. Dopamine and Serotonin Transporters DAT and SERT belong to the Neurotransmitter:Sodium Symporter (NSS) family, and serve to terminate dopamine and serotonin neurotransmission respectively, by recycling released neurotransmitters back into the presynaptic neuron. DAT is the primary target for abused psychostimulants such as cocaine and methamphetamine, whereas 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) binds to SERT. An understanding of the full spectrum of functional states and their transitions in a transporter cycle is required to understand the functions of these proteins and the complexity of the ligand binding modes, in order to identify and eventually exploit the therapeutic opportunities in reducing the efficacy of the abused drugs. Thus, the varied inhibition mechanisms of inhibitors are of particular interest in developing targeted and effective therapeutic interventions for drug abuse and other psychiatric disorders. In a recent collaborative study, a series of novel DAT inhibitors were synthesized, and based on its pharmacological profile, the lead compound was evaluated in phase I metabolic stability studies in mouse liver microsomes and compared to cocaine in locomotor activity and drug discrimination paradigms in mice. our MD simulation study supported the hypothesis that atypical DAT inhibitors have similar binding poses at DAT in a conformation that differs from that of cocaine. Such differences may ultimately contribute to their unique behavioral profiles and potential for development as cocaine use disorder therapeutics. Crystallographically, whereas both substrates and inhibitors have been found to bind in the central binding (S1) site of NSS, inhibitors were found to bind to a S2 site in the extracellular vestibule (EV) of transporters for leucine (LeuT) and serotonin. Recent binding experiments show that substrate (L-Trp) binding in the S2 site of MhsT, another bacterial NSS, is also central to the allosteric transport mechanism. We used extensive MD simulations combined with Markov state model analysis to investigate the interaction of L-Trp with the EV of MhsT and identified potential binding poses of L-Trp as well as induced conformational changes in the EV. Our computational findings were validated by experimental mutagenesis studies and shed light on the ligand binding characteristics of the EV of NSS, which may facilitate development of allosteric ligands targeting NSS. Sigma 1 Receptor The 1R is a structurally unique transmembrane protein that functions as a molecular chaperone. It is located at the mitochondria-associated endoplasmic reticulum (ER) membrane (MAM), and has been found to translocate to the plasma membrane and other parts of the cell, and modulates the functions of targets relevant to drug abuse such as dopamine receptors and DAT. Dysfunctions of such modulations are connected to many neurological disorders. In particular, 1R has been implicated in cocaine abuse. Cocaine shows biochemical affinity and pharmacological activity at both DAT and 1R. It has been demonstrated that compounds that can antagonize the action of cocaine at both sites may have therapeutic potential of cocaine abuse. 1R agonists can substitute for cocaine in self-administration, and the antagonists can block the self-administration interestingly, a few DAT inhibitors have high affinities for both DAT and 1R. However, the 1R pharmacology and the synergy of the ligands on other targets, in particular DAT, are ill-defined at molecular and cellular levels. In addition, mechanistic underpinnings of structure-function relationships of 1R are poorly understood, and molecular interactions of selective ligands with 1R have not been elucidated. We report novel bioluminescence resonance energy transfer (BRET) assays that enable analyses of ligand-induced multimerization of 1R and its interaction with BiP. Haloperidol, PD144418, and 4-PPBP enhanced 1R homomer BRET signals in a dose dependent manner, suggesting their significant effects in stabilizing 1R multimerization, whereas (+)-pentazocine and several other ligands do not. In non-denaturing gels, (+)-pentazocine significantly decreased whereas haloperidol increased the fraction of 1R multimers, consistent with the results from the homomer BRET assay. Further, BRET assays examining heteromeric 1R-BiP interaction revealed that (+)-pentazocine and haloperidol induced opposite trends of signals. From molecular modeling and simulations of 1R in complex with the tested ligands, we identified initial clues that may lead to the differed responses of 1R upon binding of structurally diverse ligands. By combining multiple in vitro pharmacological and in silico molecular biophysical methods, we propose a novel integrative approach to analyze 1R-ligand binding and its impact on interaction of 1R with client proteins.